Difructose dianhydride was first found in 1929 by Jackson et al. by analyzing a by-product which was produced when inulin was treated with sulfuric acid to prepare a fructose syrup. Difructose dianhydride, a kind of a cyclic disaccharide, consists of two fructose residues in which a reducing end of each residue is linked to a non-reducing hydroxy group of the counter residue. There have been discovered five kinds of difructose dianhydride, named DFA I to DFA V, thus far. Of them, DFA II and V are found to be synthesized only chemically while the others can be produced enzymatically: DFA I and III are produced from inulin by the action of inulin fructotransferase; and DFA IV from levan by the action of levan fructotransferase.
Difructose dianhydride is a non-digestive, non-fermentative sub-saccharide which is not digested in the animal body. In addition to being useful as a low-calorie sweetener, the sub-saccharide plays a role in inhibiting tooth decay and as a productive factor for Bifidus bacteria. It is also reported that difructose dianhydride is used as an absorption factor of minerals in the body (Baik, B. H, Lee, Y. W., and Lee, Y. B.; U.S. Pat. No. 5,700,832, UK. Pat. No. GB 2 308 547 A, Japanese Pat. Appl'n No. 8-51370, Korean Pat. Laid-Open Publication No. 96-13376, Sakurai et al., 1997).
Representative of polyfructans, inulin and levan, both naturally occurring fructose homopolysaccharides, are used to prepare difructose dianhydride (Han, 1989). From them, difructose dianhydride can be synthesized chemically or enzymatically. Chemical synthesis of difructose dianhydride from the natural polyfructans, however, suffers from significant disadvantages. For example, chemical synthesis is of low reaction selectivity, followed by complicated separation and purification. What is worse, it produces pollution of the environment. Consequently, these problems do not vest economical production value in the chemical synthesis. In contrast, enzymatic synthesis using bio-catalysts, such as microbes or enzymes, is now regarded as being very economically favorable in synthesizing difructose dianhydride from the natural polyfructans.
Since the discovery of difructose dianhydride III synthase (inulin fructotransferase) from microbes by Tanaka in 1972, enzymes that have the function of synthesizing difructose dianhydrides have been isolated from several microbial sources. The difructose dianhydrides which can be synthesized by such microbe-derived enzymes include DFA I, III and IV. DFA IV is synthesized from levan by the catalytic action of levan fructotransferase and two microorganisms, Arthrobacter ureafaciens and Arthrobacter nicotinovorans GS-9 are found to produce DFA IV.
Only a very small amount of levan fructotransferase is synthesized from these bacteria and, thus, its use in the production of difructose dianhydride is very unfavorable in terms of technical and economical aspects. Now generally, in order to obtain a large amount of a gene of interest, a gene recombinant technique is employed. That is, a levan fructotransferase gene is first isolated from its microbial source and cloned, followed by mass-expression in E. coli. Of the levan fructotransferase-producing strains discovered so far, only A. nicotinovorans GS-9 is achieved in cloning its levan fructotransferase gene.
Being a substrate of levan fructotransferase to produce DFA IV, levan, a homopolysaccharide of fructose, is prepared from sucrose by the transfructosylation reaction of levansucrase. The enzymes which can catalyze the hydrolysis of sucrose are exemplified by sucrase (beta-D-fructofuranosidase, EC 3.2.1.26), levanase (beta-2,6-D-fructan fructanohydrolase, EC 3.2.1.65), levansucrase (beta-2,6-fructan: D-glucose-1-fructosyl transferase, EC 2.4.1.10), maltase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20), etc. Levan can be produced from sucrose by taking advantage of the transfructosylation activity of levansucrase (Tanaka & Yamamoto, J. Biochem., 85, 287 (1979)).
There are disclosed methods for the production of levan using levansucrase. For example, it is described in U.S. Pat. No. 4,879,228 and International Publication No. WO 86-4091 that levan is produced by a fermentation process in which advantage is taken of the microbes employing sucrose in their metabolism. The patents, however, suffer from many problems. There is required a lengthy culture period to produce levan. Further, because the culture contains various products, a difficult purification procedure is needed, giving rise to a decrease in the production yield of levan. When account is taken of these problems, an enzyme reaction process has an advantage over the culture process in that, because the products of levansucrase are dependent on the reaction conditions for the production of levan from sucrose, desirable products can be obtained by controlling the conditions with ease.
It is reported that levan finds numerous applications in the medicinal field, such as a serum substituent (Dedonder et al., Bull. Soc. Chim. Biol., 39, 438 (1957); Schechter et al., J. Lab. Clin. Med., 61, 962(1963)), a colloid stabilizer, an immune agent, a pharmacological enhancer, etc. (Leibovici et al., Anticancer Res., 5, 553 (1985); Stark et al., Br. J. Exp. Path., 67, 141 (1986)), the foodstuffs field, such as quality improver, a stabilizer, an additive for health food, etc. (Hatcher et al., Bioprocess, Technol., 11, 1(1989)), the typographic field, and the cosmetic field (Whiting et al., J. Inst. Brew., 73, 422 (1967); Han, Bioprocess Technol., 12, 1(1920)).